Studies on the Metabolic Control of Caffeine Turnover in Developing

Leaves and immature fruits of Coffea dewevrei and Coffea arabica var. Mundo Novo and var. Laurina were fed with [8-3H] caffeine and the products analy...
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J. Agric. Food Chem. 1994, 42, 7423-7427

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Studies on the Metabolic Control of Caffeine Turnover in Developing Endosperms and Leaves of Coffea arabica and Coffea dewevrei Paulo Mazzafera,*p+Alan Crozier,$ and Goran Sandbergs Departamento de Fisiologia Vegetal, IB, Universidade de Campinas, CP 6109, 13081-970 Campinas, SP, Brazil, Department of Botany, School of Biological Sciences, The University, Glasgow G12 SQQ, U.K., and Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Science, S 90183 Umei, Sweden

Leaves and immature fruits of Coffea deweurei and Coffea arabica var. Mundo Novo and var. Laurina were fed with [8-3Hlcaffeine and the products analyzed by reversed-phase high-performance liquid chromatography with ultraviolet and radioactivity detection. In fruits, only endosperms were analyzed. In addition, the activities of two methyltransferases involved in the biosynthesis of caffeine were studied in cell-free preparations from leaves and endosperms. The results provide evidence that the low caffeine content in leaves and endosperms of immature fruits of C. deweurei may be due to slow biosynthesis and fast degradation. The high caffeine content in leaves and endosperms of C. arabica seems to be a consequence of a rapid rate of production coupled with a slow degradation rate.

INTRODUCTION Caffeine (1,3,7-trimethylxanthine) is the major alkaloid in coffee, and variations in caffeine content are found within the same tree, with the highest levels in seeds, flowers, and leaves (Raju and Gopal, 1979). Variability is also observed among varieties of the same species and among different species of coffee (Charrier, 1978; Mazzafera and Carvalho, 1992). Studies on the synthesis and degradation of caffeine in coffee have been carried out, predominantly, with fruits of Coffea arabica [for a review see, Suzuki et al. (199211, and although the metabolic steps are known (Figure 11, the control of the level of caffeine in coffee tissues is not fully understood. Adenine and guanine, derived from the nucleotide pool, are the primary precursors of caffeine in coffee (Suzuki and Waller, 1984b). Thus, its level, in some tissues, might be related to general metabolic activity (Waller and Nowacki, 1978), and tissues such as young leaves, flowers, and immature fruits should be expected to contain more caffeine than mature or old tissues (Clifford and Kazi, 1987; Frischknecht et al., 1986; Raju and Gopal, 1979). Roberts and Waller (1979) and Suzuki and Waller (1984a,b)demonstrated that immature fruits had a higher capacity for caffeine degradation, even though they contained more caffeine than mature fruits. A t the same time, cell-free preparations from immature fruits had higher activities of methyltransferases involved in the biosynthesis of caffeine than preparations from older fruits (Roberts and Waller, 1979). These investigations described differences found in fruits of C. arabica a t different stages of development but did not deal with differences between species or different tissues of the same tree. In a previous paper, we showed that endosperms of immature fruits of different coffee species markedly differed in their catabolism of caffeine (Mazzafera et al., 1991). Endosperms from Coffea deweurei, containing 0.25 ?4 caffeine, had a much higher degradation rate than

* Author to whom correspondence should be addressed. + Universidade de Campinas. t

5

The University, Glasgow. The Swedish University of Agricultural Science. 002 1-856 7 19411442-1423$04.5QIO

those from C. arabica var. Mundo Novo, having 1.3% caffeine. The caffeine contents in endosperms of mature fruits of these species were 1.2% and 1.1% , respectively. It was suggested, therefore, that the turnover of caffeine is high in endosperms of immature fruits of C. dewevrei, decreasing with maturation. Caffeine contents in endosperms from immature and mature fruits of C. arabica var. Laurina were 0.8 % and 0.6 % ,respectively, although showing the same degradation rate as C. arabica var. Mundo Novo (Mazzafera et al., 1991). The difference in caffeine content between these two C. arabica varieties might be due a higher biosynthesis rate in var. Mundo Novo. In this paper we examine the results of in vitro and in uiuo experiments, performed to study the synthesis and degradation of caffeine in leaves and endosperms of immature fruits of C. deweurei and C. arabica var. Mundo Novo and var. Laurina. The activities of N-methyltransfemes, catalyzing the methylation of 7-methylxanthine and theobromine (3,7-dimethylxanthine), were assayed in cell-free preparations of leaves and endosperms of immature fruits. In the in vivo experiments, leaves and immature fruits were fed with [8-3H]caffeine before extraction and analysis of metabolites by reversed-phase high-performance liquid chromatography (RP-HPLC), with ultraviolet and radioactivity detection. MATERIALS AND METHODS Radiochemicals. [€b3H]Caffeine (22.2 Ci/mmol) and Sadenosyl[3H-CH3]methionine(15Ci/mmol)were purchased from Amersham International (Sweden). In Vivo Experiments. Plant Material. Immature fruits of C. deweurei (30 weeks old) and of C. arabica var. Mundo Novo and var. Laurina (25weeks old) were collected at the Experimental Station of Instituto Agronbmico, Campinas, SP, Brazil, between August 1991 and January 1992. Leaves of the third pair were collected in November 1991. Fruits and leaves were collected from the same coffee trees used by Mazzafera et al. (1991). The C. deweurei tree was 50 years old, and both C. arabica trees were 8 years old. Radioisotopic Feeding Experiments. Radioisotopes were fed to fruits essentially as described by Mazzafera et al. (1991). Each fruit received 2 pCi of [8-3H]caffeineor 2 pCi of [S-SHIcaffeine plus 50 pg of theophylline (1,3-dimethylxanthine).Theophylline was dissolved in 50 mM sodium phosphate, pH 6.0, and 0.65 mM 0 1994 Amerlcan Chemlcal Soclety

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O

N I

CII, THEOBROt4INE (3,7-DlMETHYLXANTHINE)

2

ICsAM THEOPKTUNE

(1,3-DIMETHYLXANTHlNE) 1( #’

k

CH, MoBcrxvJE

7METHYU[ANTHINE

(3.7-DIMETHYLXANTHINE)

“TcC ACID

C%

+ NH3

Figure 1. Biosynthesis and biodegradation pathways of caffeine in coffee: 1, 3-N-methyltransferase; 2, 1-N-methyltransferase; *, SH-label in the caffeine molecule. ascorbicacid. After 12h, the fruitswere cut and the white opaque endosperms collected, dried at 80 OC, and finely ground in a mortar. Endosperms from at least 35 fruita were used in each treatment. For radioisotopic feeding of leaves, whole branches were collected and washed thoroughly with tap water. After quick blotting with absorbent tissue, leaves of the second and third pairs were detached and the petiole was immersed in vials containing 1.5 mL of 50 mM sodium phosphate, pH 6.0,0.65 mM ascorbic acid, and 1.5 pCi of [&aHlcaffeine with or without 800 pg of theophylline. During the incubation period, any solution absorbed by the leaves was replaced by buffer. After 24 h under constant light (300 pmol m-2 8-1) at 25 & 2 O C , the petioles were quickly washed under tap water, and the leaves were dried at 80 O C and finely ground. The volumes of the remaining solutions in the vials were determined, and aliquots were removed to measure the uptake of [8-SHlcaffeineand theophylline. [8JHICaffeine was measured by radiometry in a scintillation counter and theophylline by RP-HPLC at 280 nm. The column used was a TSK ODs-12OT (Pharmacia;5 wm, 250 mm X 4.6 mm id.), and chromatography was developed isocratically with 35%MeOH in distilled H20 at a flow of 1mL/min. Ten leaves were used in each treatment. Extraction of Caffeine and Metabolites. Leaves and endosperms were extracted with 6.3 mM H a 0 1 according to the method of Petermann and Baumann (1983) and the extracts clarified by addition of heavy MgO. After centrifugation, the supernatants were freeze-dried, resuspended in 2 mL of distilled H*O, and passed through CIS Sep-Pak cartridges (Millipore, Waters). The first eluate (xanthine was poorly retained) and subsequent washings with 2 X 2 mL of 50% MeOH were combined, freeze-dried, resuspended in 0.4 mL of distilled H20,

and filtered into Ultrafree-MC Eppendorf tubes (Millipore, Waters). These extracts were used for the determination of endogenous methylxanthmes, [&sH]caffeine, and metabolites by RP-HPLC. Two replicates of extraction and further analysis in RP-HPLC were made for each treatment. High-Performance Liquid Chromatography. A CIS guard column and an ODS Hypersil column (5 pm, 250 mm X 4.6 mm i.d.1 (Capital HPLC Specialist, U.K.) were used with a gradient of 30 min of 0 4 5 % MeOH in 0.5% acetic acid, pH 5.0 (with 5 M NaOH),and a flow rate of 1mL/min. The elution was followed at 280 nm, and when labeled samples were analyzed, the eluate emerging from the absorbance monitor was mixed with scintillation fluid and monitored by a radioactivity monitor, as described by Mazzafera et al. (1991). In Vitro Experiments. Plant Material. Fruits used here were collected simultaneously with those used in the in vivo experiments. They were washed with tap and distilled water before being cut into halves. The endosperms were removed and immediately immersed in 100 mM sodium phosphate, pH 7.0,and 6.5 mM ascorbic acid at 4 OC. The buffer was drained, and the endosperms were frozen at -20 OC, freeze-dried, finely ground in a mortar, and stored at 4 OC until use. Leaves of the second pair were collected from 12-month-old seedlings grown under greenhouseconditions (18/6h, 24/21 “C)in UmeH, Sweden, in July 1992. They were frozen in liquid nitrogen, freeze-dried, finely ground, and stored at 4 OC until use. Cell-Free Reparations and Enzyme Assays. Leaves from two plants composed one replicate, and four such replicates were used for each coffee species or variety. As fruits were collected from individual trees, four independent extractions and measurements of activities were made. Freeze-dried leaves and endosperms were extracted with a buffer modified from that of

Metabolic Control of Caffeine Turnover Wingsle et al. (1991) containing 50 mM sodium phosphate, pH 7.5, 5 mM DTT, 5 mM EDTA, 6.5 mM ascorbic acid, and 4% poly(vinylpyrro1idone)(M, 360 OOO) in a proportion of 0.1 g/mL. After 15 min on ice, 0.25 mL of cold saturated aqueous (NH& SO4 solution was added per milliliter of extract. The gummy precipitate was eliminated by centrifugation at 31OOOg for 30 min at 4 O C . The supernatant was filtered through Sephadex PD-10 columns (Pharmacia) and eluted with 50 mM sodium phosphate, pH 7.5. The filtrate was used for measurements of N-methyltransferase activity. These were carried out for 30 min at 28 O C in Eppendorf tubes in reaction mixtures containing 50 mM sodium phosphate, pH 7.5,0.75 mM substrate (theobromine or 7-methylxanthine1, 3 mM MgC12, ca. 100 pg of protein, and 0.1 pCi of S-adenosyl[3H-CHs]methionine, as the methyl donor, in a final volume of 100 pL. Our previous trials had shown that, under the conditions described above, enzyme activities were linear up to 13 min, with highest activities being attained at 15 min, and a longer incubation period (30 min) gave similar final activities. Good proportionality was observed up to 120 pg of protein added to the reaction mixture. The reaction was started methionine and stopped with the addition of S-adeno~yl[~H-CH31 with 10 pL of 6 M HCl. After addition of 1 mL of chloroform, the Eppendorf tubes were vortexed for 2 min and briefly centrifuged for separation of the phases. RP-HPLC showed that when theobromine was used as substrate, S-aden0syl[~H-CH3]methionine was retained in the aqueous phase and the product [3H-CH3]caffeine transferred to the chloroform phase. The organic phase was dried in scintillation vials, and the radioactivity was counted after addition of scintillation fluid. When 7-methylxanthine was used as substrate, the reaction was stopped with 100 p L of acetone, -20 OC,and the tube was centrifuged to eliminate precipitated proteins. Aliquots from the supernatant were dried under a stream of nitrogen, resuspended in distilled water, and analyzed by RP-HPLC,as described for the metabolites of [8-3H]caffeinefor the in uiuo experiments. In the assay for 3-N-methyltransferase only theobromine could be detected in the chromatograms. Protein Determination. Protein was assayed according to the method of Bradford (1976) using a prepared Bio-Rad reagent. RESULTS AND DISCUSSION After anthesis, fruit maturation in C . arabica takes ca. 32 weeks and in C.dewevrei ca. 50 weeks. T o obtain fruits a t a similar stage of development, fruits of C. arabica and C . dewevrei were collected a t 25 and 30 weeksafter anthesis [cf. Mazzafera (1990)l. At the time of collection the dry matter contents of fruits of C . arabica var. Mundo Novo and var. Laurina and C . dewevrei were 21.1 5% ,19.5 % ,and 20 % , respectively, and endosperms represented 3.4 % , 3.1 %, and 2.8% of the total dry matter. Estimates of endogenous contents of caffeine and related methylxanthines (Table 1) are in agreement with data reported in the literature (Fob6 and Carvalho, 1965; Mazzafera et al., 1991; Raju and Gopal, 1979). Caffeine was the alkaloid present in higher amounts in endosperms and leaves of both species. Endosperms of C. dewevrei also showed a much higher theobromine content. In the radioisotopic feeding experiments the uptake of caffeine and theophylline by leaves was determined. Both C. arabica varieties absorbed ca. 65% of [8-3Hlcaffeine, and 80% was absorbed by C . dewevrei. When supplied together, ca. 30% of theophylline and ca. 35% of [fb3H1caffeine were absorbed by both C . arabica varieties and 65 % and 66 5% ,respectively, by C. dewevrei. An increase of theophylline content was detected in leaves and endosperms; however, the endogenous contents of caffeine and theobromine were not affected (Table 1). As previously observed (Mazzafera et al., 1991), for all treatments there was a low recovery of the applied radioactivity (Table 2). For fruits this would be partially explained because only endosperms were analyzed. Nevertheless, extraction with [8-3Hlcaffeine blanks also gave

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Table 1. Content of Methylxanthines in Endosperms of Immature Fruit and Leaves of C.arabica Var. Mundo Novo and Laurina and C. dewevrei, As Determined by RP-HPLC and UV Detection m d a drv weinhta treatment species theobromine theophylline caffeine ~

~~

Endosperms 3H-~af

M.Novo Laurina C. dew.

3H-caf + Thp M. NOVO Laurina C. dew.

0.040 0.042 0.367

0.003 0.001 0.007

12.6 6.7 4.7

0.033 0.039 0.334

0.027 0.048 0.051

11.7 7.7 4.3

Leaves

3H-~af

M.Novo Laurina C. dew.

3H-caf + Thp

M. NOVO Laurina C. dew.

0.103 0.035 0.020 0.087 0.040 0.027

ND

9.3

ND 0.005

7.3 0.2

0.197 0.209 0.098

8.6 7.1 0.2

a Data are means of two replicates and in no case was the coefficient of variation higher than 8%. 3H-caf, [8-*H]caffeine;Thp, theophylline; 3H-caf + Thp, [8-3H]caffeineplus theophylline; M. Novo, Mundo Novo; C.dew., C.deweurei; ND, not detected.

low yields (45-50% recovery), indicating losses due to adsorption during the extraction and purification procedures. In agreement with earlier studies (Mazzafera et al., 1991), fruits of var. Mundo Novo and var. Laurina did not metabolize caffeine as rapidly as C. dewevrei fruits, and the same was observed in leaves (Table 2). In keeping with data obtained with C. arabica varieties, less radioactivity was recovered from endosperms and leaves of C. dewevrei, and products of caffeine catabolism accumulated radioactivity in this species. Two polar compounds (I and 11),detected previously by Mazzafera et al. (1991)in fruits, were also detected in leaves, and their radioactivities are shown in Table 2. The identities of these compounds are under investigation since they can be catabolites of an alternative caffeine degradation pathway. Romero and Waller (1988) isolated from coffee fruits a compound that was identified as a product of theophylline catabolism. In coffee fruits caffeine is mainly synthesized via theobromine and degraded via theophylline (Suzuki and Waller, 1984a,b). Although feeding experiments have shown that theophylline could be also involved in the biosynthesis of caffeine in fruits of C.arabica, considering theobromine as reference, only 4% of the applikd theophylline was converted into caffeine. In such biosynthesis theophylline would be synthesized by the methylation of 1-methylxanthine(Suzuki and Waller, 1984a,b). However, since caffeine degradation involves two distinct N-demethylations (cf. Figure l),the theobromine content in endosperms of C. dewevrei might be a result of a more active N-demethylating step rather than a consequence of higher caffeine degradation rate. As theobromine is the immediate precursor as well as one of the products of caffeine catabolism, experiments with labeled theobromine can lead to erroneous conclusions. Thus, fruits and leaves were fed with [€b3H1caffeine plus theophylline with the expectation that 7-N-demethylation of caffeine to theophylline would be inhibited and caffeine catabolism converted to theobromine, via 1-N-demethylation (Table 2). It was observed, however, that inhibition of [8-3Hlcaffeine conversion was higher in endosperms of C. dewevrei for the theobromine than for the theophylline route. These findings suggest an effective conversion of caffeine into theobromine in this coffee species.

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Table 2. [8-*H]Caffeine Metabolites in Endosperms of Immature Fruit and Leaves of C. arabica Var. Mundo Novo and Laurina and C. dewevmi content (7% of recovered radioactivity) treatment species recovered' (% ) 1/11 Xan 3mx Thb ThP Caf Endosperms 3H-caf M. Novob 23.5 0.49 ND ND 0.12 ND 99 Laurina 20.2 0.45 ND ND 0.32 ND 99 C. dew. 14.1 1.03 0.82 1.97 35.0 2.44 59 3H-caf + Thp M. Novo 20.3 0.44 ND ND 0.26 ND 99 Laurina 22.7 0.22 ND ND ND ND 99 C. dew. 13.2 0.85 ND 0.21 10.8 2.27 86 Leaves M. Novo 19.9 ND ND ND ND ND 100 ND ND ND ND 100 Laurina 13.2 ND C. dew. 4.7 10.0 ND 4.54 7.1 15.8 63 3H-caf + Thp M. Novo 34.0 ND ND ND ND ND 100 ND ND ND ND ND 100 Laurina 30.5 C. dew. 23.9 2.72 ND 1.93 3.5 7.2 85 Data are expressed as percentage of applied radioactivity (2 pCi/fruit). Data are means of two replicates, and in no caee was the coefficient of variation higher than 11%.1/11, compoundsI and II; Xan,xanthine; 3mx, 3-methylxanthine; Thb, theobromine; Thp, theophylline; Caf, caffeine; SH-caf,[&SH]caffeine;aH-caf+ Thp, [&SH]caffeine plus theophylline; M. Novo, Mundo Novo; C. dew., C. deweurei;ND,not detected. 3H-caf

Table 3. Activities of 1-N-Methyltranaferane and 3-KMethyltransferane in Endosperms of Immature Fruit and Leaves of C. arabica Var. Mundo Novo and Laurina and C. dewevrei endosperms' leaves' species 1-N 3-N 1-N 3-N 7.1k0.4 5.1f0.7 4.5a0.8 MundoNovo 5.9f0.3 6.8 f 0.9 4.2 f 0.8 2.6 f 0.6 3.1 f 0.2 Laurina 1.4 f 0.3 ND 0.9 f 0.4 ND C. deweurei 0 Data are expressed as picomoles of [VH]theobromineproduced by milligram of protein for 3-N-methyltrannferaseand picomoles of [@H]caffeine produced by milligram of protein for 1-N-methyltransferase. 1-N, 1-N-methyltransferassactivity; 3-N, 3-N-methyltransferase activity; ND, activitynot detected;means f SD of four replicates. Theophylline affected [8-3H]caffeine catabolism in leaves of C. dewevrei in a different ways (Table 2). Both theophylline and theobromine routes seemed to be inhibited to ca. 50 %. Radioactivity in 3-methylxanthine also decreased aproximately 50 5%. A possible explanation for the effects of theophylline addition in the [8-3Hlcaffeine catabolism in C. dewevrei could be that both N-demethylating reactions are catalyzed by the same enzyme but show different affinities for the methyl groups in the caffeine molecule. This has been observed in bacteria (Dixon and Webb, 1979). Petermann and Baumann (1983) showed that, in a specific developmental stage, young leaves of C. deweurei can also degrade caffeine through methyluric acids. We could not detect endogenous methyluric acids in our material; however, since the tritium label of [8-3Hlcaffeine is lost during oxidation to uric acid derivatives, we cannot exclude the contribution of this degradative pathway occurring in the degradation pathway of [8-3Hlcaffeine (Figure 1). Table 3 presents the results of the enzyme assays for the 1-N- and 3-N-methyltransferase activities in endosperms and leaves. Lowest activities were found in C. dewevrei. Endosperms of C.arabica var. Mundo Novo and var. Laurina showed similar activities of 1-N- and 3-N-methyltransferases, and excluding 3-N-methyltransferase activity in leaves of var. Mundo Novo, endosperms had higher activities than leaves. The activities of both N-methyltransferases were higher in leaves of var. Mundo Novo.

We did not study other enzymes of caffeine biosynthesis, but the results of the 1-N- and 3-N-methyltransferase assays and the data from the radioisotopic feeding experiments suggest that a high degradation rate combined with a low biosynthesis rate may explain the low content of caffeine in leaves and endosperms of C. dewevrei (see Table 1). On the other hand, the reverse situation occurs with C. arabica-a slow degradation rate combined with a high rate of synthesis. However, these data do not provide a satisfactory explanation for the differences in the caffeine contents between the varieties Laurina and Mundo Novo. Since mature endospermsof C. dewevrei contain as much as 1.2% caffeine, one might expect significant alterations in the rate of biosynthesis and biodegradation of caffeine during fruit ripening. As endosperms of immature and mature fruit of C. arabica have similar levels of caffeine, such a pronounced alteration would not be expectad during fruit ripening. ACKNOWLEDGMENT We thank Gunnar Wingsle and Ladaslav Sodek for critical reading of the manuscript. P.M. thanks CNPqBrazil for granting a fellowship during a leave of absence a t The Swedish University of Agricultural Science, UmeA, Sweden. LITERATURE CITED Bradford, M. N. A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. Charrier, A. La structure ghnhtique des cafbiers spontanb de la r6gion Malgache (Mascarocoffea). Leurs relations auec les cafkiers d'origine africaine (Eucoffea); MBmoires ORSTOM 87; ORSTOM: Paris, 1978. Clifford, M. N.; Kazi, T. The influence of coffee bean maturity on the content of chlorogenic acids, caffeine and trigonelline. Food Chem. 1987,26,59-69. Dixon, M.; Webb, E. C. In Enzymes; Longman Group: London, 1979; p 1116. FobQ L. A.; Carvalho, A. Caffeine content in different parts of coffeetrees in correlation with genetic characteristics. In First Session, Technical Working Party on Coffee Production and Protection; Minist6rio da Indfmtria e Combrcio e Instituto Brasileiro do cafB: 1965; p 1. Frischknecht, P. M.; Ulmer-Dufek, J.; Baumann, T. W.Purine alkaloid formation in buds and developing leafleta of Coffea

Metabolic Control of Caffeine Turnover arabica: Expression of an optimal defence strategy? Phytochemistry 1986,25,613-616. Mazzafera, P. Metabolism of Caffeine in CoffeaL. Ph.D. thesis, Universidade de Campinas, Campinas, Brazil, 1990. Mazzafera,P.; Carvalho,A. Breedingfor low seed caffeinecontent of coffee (CoffeaL.) by interspecific hybridization. Euphytica 1992,59,55-60. Mazzafera,P.; Crozier,A,;Magalhiies,A. C. Caffeinemetabolism in Coffea arabica and other species of coffee. Phytochemistry 1991,30,3913-3916. Petermann, J. B.; Baumann, T. W. Metabolic relations between methylxanthines and methyluric acids in Coffea L. Plant Physiol. 1983, 73,961-964. Raju, K.1.; Gopal, N. H. Distribution of caffeine in arabica and robusta coffee plants. J. Coffee Res. 1979,9,83-90. Roberta, M. F.; Waller,G. R. N-methyltransferases and 7-methylNe-nucleoside hydrolase activity in Coffea arabica and the biosynthesis of caffeine. Phytochemistry 1979, 18,451-455. Romero, I. C. L.; Waller, G. R. Production of a new compound by metabolism of theophylline in Coffea arabica L. Rev. Latinoam. Quim. 1988,19,46-50.

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Suzuki, T.; Waller, G. R. Biodegradation of caffeine: formation of theophylline and theobromine from caffeine in mature Coffea arabica fruits. J. Sci. Food Agric. 1984a, 35, 66-70. Suzuki, T.;Waller, G. R. Biosynthesis and Biodegradation of caffeine, theobromine, and theophylline in Coffea arabica L. fruits. J . Sci. Food Agric. 1984b,32,845-848. Suzuki,T.; Ashihara, H.; Waller,G. R. Purine and purine alkaloid metabolism in Camellia and Coffea plants. Phytochemistry 1992,31,2575-2584. Waller,G. R.; Nowacki, E. K. In Alkaloid Biology and Metabolism in Plants; Plenum Press: New York, 1978;p 293. Wingsle, G.; Gardestrijm, P.; Hiillgren, J.-E.; Karpinski, S. Isolation, purification, and subcellular localizationof isozymes of superoxide dismutase from Scots Pine (Pinus sylvestris L.) needles. Plant Physiol. 1991,95,21-28. Received for review September 13,1993. Revised manuscript received February 24, 1994. Accepted March 23,1994.@ 0 Abstract

1, 1994.

published in Advance ACS Abstracts, May